SK151199A3 - Pharmaceutical compositions of tizoxanide and nitazoxanide - Google Patents

Abstract

A pharmaceutical composition containing as active agent at least one compound selected among the group consisting of formula (I) and formula (II) and further containing at least one pharmaceutically acceptable acid.

The active agent is preferably in the form of particles having a particle size smaller than 200 µm and a mean particle size of greater than 10 µm. The pharmaceutical compositions according to the invention are stabilized with at least one pharmaceutically acceptable acid. The pharmaceutical compositions are particularly useful for treatment of opportunistic infections in persons with compromised or suppressed immune systems, and in treatment of infections of trematodes.

Description

Technical field

The present invention relates to a pharmaceutical composition comprising as an active ingredient at least one compound selected from the group consisting of a compound of formula (I)

and a compound of formula (II)

The active ingredient is preferably in the form of particles below 200 μιη and the mean particle size is greater than 10 μιη.

The invention also relates to pharmaceutical compositions stabilized by at least one pharmaceutically acceptable acid.

The pharmaceutical compositions are particularly useful in the treatment of opportunistic

I, infections of individuals with a weakened or suppressed immune system and in the treatment of infections with fluke (trematodes).

BACKGROUND OF THE INVENTION

There is an urgent need to develop methods for treating numerous parasitic and bacterial infections of immunocompromised persons (AIDS, carcinoma, the elderly, aging diseases, recipients of immunosuppressive drugs after organ transplantation). Another area of interest is fluke infections, especially in tropical climates. Accordingly, there is a need for a pharmaceutical composition that is also tolerated by immune-compromised individuals and that can be stored in a tropical environment.

Specifically, the element Toxoplasma gondii is among the predominant causes of latent central nervous system infections worldwide. Many healthy people are infected with this parasite, but usually their immune system keeps the organism under control. Toxoplasma gondii is the most common opportunistic brain pathogen of AIDS patients. Nowadays, toxoplasmosis is becoming an increasing problem not only because of AIDS, but also due to the increasing use of immunosuppressive drugs (administered, for example, to organ transplant patients). Toxoplasmosis is usually treated with a combination of pyrimethamine and sulfadiazine. They are effective drugs, but they do not kill the parasite cysts, so treatment must be continued at maintenance doses. Toxicity often necessitates discontinuation of the drug, especially in people immunosuppressed (receiving immunosuppressive drugs), followed by recurrence. The statistics are therefore not favorable at the reported mortality of about 70% in immunodeficient patients and a mean survival of four months.

Cryptosporidiosis is caused by the microscopic parasitic element Cryptosporidium parvum. In persons with normal immunity, diarrhea caused by this element can be severe and long but self-limiting. Cryptosporidial diarrhea often threatens their lives in AIDS patients. It is estimated that about 15-20% of AIDS patients suffer from this disease. To date, there has been no sustained or effective therapy for cryptosporidiosis.

In AIDS patients, Enlerocylozoon hietieusi was most commonly identified as a pathogen, a parasitic microsporid found in almost one quarter of patients. There is no effective treatment to date.

HIV-positive persons infect several other types of microsporidia including Encephalilozoon helium and cuniculi and a new species designated as Whispera intestinalis. A recent report finds that disseminated microsporidial infection is currently gaining in importance.

Isospora heHi is a clinically indistinguishable infection from cryptosporidiosis. It is known earlier in the tropical zone and has been found in less than 1% of patients in the US, although its actual incidence is likely to be higher.

Pnenmocystis carinii is generally classified as a parasitic element; however, some reports suggest that it could be one of the fungi with which it shares certain genetic sequences. Pnenmocystis carinii usually infects the lungs (Pneumocystis Carinii Pneumonia (PCP) - pneumonia caused by P.c.). Treatment is reported to be successful in 40-60% of patients, but there are problems with drug toxicity especially in immunocompromised patients. Among many serious cases of human immunodeficiency virus (HIV) infection in children, PCP has a special position due to its high incidence, rare age distribution, and frequent mortality. PCP is the most common serious opportunistic infection in HIV-infected children; the incidence of PCP among young HIV infected children without prophylactic treatment is estimated to be at least 12% in the first year of life. Many children die shortly after the development of PCP.

Mycobacterium Avium Complex (MAC) belongs to infections caused by a family of very similar mycobacterial microorganisms Mycobacterium avium and Mycobacterium inlracellnlare. If it occurs in people not immune-compromised, it is usually in the form of respiratory tract infection. In patients suffering from AIDS, MAC is usually disseminated (disseminated MAC or □ MAC) and almost any organ can be affected. A recent study found that MACs were found in 43% of patients who survived 2 years after AIDS was diagnosed. No standard therapy was found for disseminated MAC. Combinations of medicines are usually prescribed and, if successful, require life-long administration. There is an urgent need for a more effective treatment.

People infected with HIV are particularly susceptible to Mycobaclerium tuberculosis infections and the course of the disease is accelerated. While extrapulmonary tuberculosis is uncommon in people not infected with HIV, it is common in HIV-positive people. The CDC published guidelines for the treatment of TB that take into account the growing incidence of drug-resistant tuberculosis (MDR-TB). Mortality among AIDS patients with MDR-TB is very high (about 80%) and the progression of the disease is extremely severe.

Therefore, there is an urgent need to develop new treatments for these infections, which are so common in humans and animals and endanger them permanently.

There is also a need for broad spectrum drugs to facilitate the treatment of trematode infections. Currently, it is necessary to diagnose a specific pathogenic fluke and prescribe fluke-specific drug therapy. Many less developed countries do not have the means to diagnose specific fluke. Finding a broad-range medicine would eliminate the need for this diagnosis.

Schistosoma mansoni, blood fluk, is the cause of schistosomiasis, the second most important tropical parasitic disease in humans after malaria, and the most important flukes infection in humans. Schistosoma haematohium is another important species infecting humans. Worldwide, more than 200 million people, including several hundred thousand people in the US, suffer from schistosomiasis.

Fasciola hepatica, a common liver fluk, is primarily a disease of sheep, but man is a random host. This parasite can survive even with a strong host immune response. Bithionol is offered for treatment but is not approved in the United States.

Therefore, there remains a need for a pharmaceutical composition with a long shelf life, even under tropical conditions and with a broad anti-fluke action.

SUMMARY OF THE INVENTION

It has now been observed in animal and clinical human studies that the efficacy of treatment with the compounds of formula (I) and (II) depends on the particle size of the active ingredient of the drug and the stability of the compound.

The disclosed pharmaceutical compositions are suitable for the treatment of human and animal fluke caused by Schislosoma pathogens such as Schislosoma mausoni, Schislosoma haemalohium, Schislosoma mekongi, Schislosoma japonicum, Schislosoma intercalatum; Fasciola pathogens such as Fascioia hepalica and Fasciola gigantica, Fasciolopsis hiski; and Dicrocoelium dendrilicum, Helerophyes heterophyes and Metagonimus yokogawa.

The pharmaceutical compositions are also effective in the treatment of immuno-compromised patients suffering from opportunistic infections with the pathogens Cryptosporidium parvum, Isospora belli, Enlerocylzoon bineusi, Encephalitozoon intestinalis, Mycobacterium tuberculosis, Mycobaderium avium intracellulare, Pnenmocyslis carcinoma, Pnenmocyslis.

The pharmaceutical composition may be in a form suitable for oral administration, as a solid dosage form, a liquid suspension or as a paste.

For a better understanding of the nature and object of the invention, it would be useful to refer to the following detailed description, which should be read with the accompanying drawings in which:

Figure 1: shows percent inhibition and survival of host cells when nitazoxanide was administered against E. intesinalis.

Figure 2: shows percent inhibition and survival of host cells when nitazoxanide was administered against Iβ. corneae.

Figure 3: Shows percent inhibition and survival of host cells when administered with albendazole against E. intestinalis.

Figure 4 shows percent inhibition and host cell viability of albendazole against EXAMPLE ⁷ corneae.

Figure 5 and 6: shows the optical density values obtained for each culture well plotted against drug concentrations in culture.

Figure 7 is a graph based on the determination of nitazoxanide activity against mycobacteria propagated in liquid nutrient broth.

Figure 8 shows the percentage of active particles with a volume of less than 0 µm.

The method of treating infections of the invention comprises administering a pharmaceutical composition comprising as an active ingredient at least one compound selected from the group consisting of the desacetylnitazoxanide of formula (I)

and the nitazoxanide of formula (II)

Nitazoxanide (NTZ), a compound of formula (U), is the generic name of 2 (acetyloxy) -N- (5-nitro-2-thiazolyl) benzamide, a compound which was first synthesized by Rossignol and Cavier in 2003. 1975 2 mg of nitazoxanide can be dissolved in 1 ml of DMSO. Nitazoxanide is readily absorbed by oral administration

Ί

To date, there has been no evidence that compounds of formula (I) and / or (II) could be broadly effective against fluke infections or that they would be sufficiently nontoxic to be tolerated by immuno-attenuated persons.

The preparation and certain uses for nitazoxanide are described in U.S. Patent 3,950,351 and in the publications published by the Applicant. Desacetylnitazoxanide, a compound of formula (1), is sometimes referred to as tizoxanide or d-NTZ and is a metabolite of nitazoxanide.

In WO 95/28393 the Applicant has described a process for preparing a pure compound of formula (II) as well as the use of a composition comprising a mixture of compounds of formula (I) and (Π).

It has now been observed that solid particles of a compound of formula (I), a compound of formula (II) or mixtures thereof with a particle size between 170 and 520 gm (mean particle size = 352 gm) have very limited efficacy when administered orally to humans or animals. The efficacy of such particles is less than that of existing pharmaceutical products and is therefore unacceptable for commercial purposes.

In dogs, it was also observed that oral administration of a single dose of 50 mg solid particles of a compound of formula (I) and a compound of formula (II) with a particle size below 5 μιη per kg of dog weight caused severe adverse effects.

It has now been found that, in order to ensure effective and safe treatment of infections caused by humans and animals with parasites, bacteria, fungi and viruses, the pharmaceutical composition, whether in solid dosage form or liquid suspension, must contain an effective dose of the active ingredient in solid particulate compound. of formula (1) and / or formula (II) having a particle size of less than 200 μιη, the mean particle size of the active solid particles being greater than 10 μηι.

The presence of a high particle content of the active ingredient larger than 200 μιη relative to a particle size between 5 and 200 μπι significantly reduces the chemotherapeutic activity of the compounds. It is preferred that the pharmaceutical compositions of the invention contain no more than 5% by weight of the active solid particles of greater than 200 microns. Most preferably, the pharmaceutical compositions of the invention contain practically no active solid particles of greater than 200 microns in size.

The presence of a high active ingredient particle size below 5 μιη relative to a particle size between 5 and 200 μιη may produce negative effects in animals or humans. In addition, particles with dimensions below 5 μιη have been found to be absorbed more rapidly from the gastrointestinal tract into the bloodstream and are therefore not sufficiently effective against parasites, bacteria, fungi and viruses in the digestive tract of animals and humans.

Even experienced scientists could not predict that the particle size of the compound of formula (I) and the compound of formula (II) could have such a significant impact on their antimicrobial activity in the treatment of animals and humans. For example, in the investigations conducted by the applicant, antiparasitic compounds such as albendazole, mebendazole, niclosamide, praziquantel and metronidazole have not shown such a significant difference in antiparasitic activity in the treatment of animals and humans depending on their particle size. In addition, even an experienced scientist could not predict that the particle sizes of the compound of formula (I) and the compound of formula (II) would have such an adverse effect on the ability of animals and humans to tolerate administration of said active ingredient.

The compounds of formula (I) and (II) may be administered either as a solid dosage form or as an aqueous suspension, but it is preferred that the pharmaceutical compositions contain an effective dose of the active ingredient in the form of a solid particle of formula (I) and / or (II) particle size below 200 μιη, the mean particle size of said active solid being greater than 10 μιη as can be detected with a Coulter Counter LS 100. This device uses 750 nm laser beams to classify particles in the range of 0.4 μιη to 900 μιη (diameter) Refraction The samples are measured in water with little addition of Triton X-100 to increase wettability and deflocculate the powder.

It is preferred that the mean particle size of said active solid particles is between 10 and 100 µm, preferably between 20 and 50 µm. Examples of preferred compositions are:

- compositions with less than 10% by weight of said active solid particles with a particle size above 100 μηι;

- compositions with at least 50% by weight of said active solid particles with a particle size below 50 μπι;

Preferably, the mean particle size of said active solid particles is between 10 and 100 μη, more preferably between 20 and 50 μη. In a preferred embodiment of the composition, less than 10% of said active solid particles have a particle size of less than 5 μη.

The active ingredient (s) used in the solid dosage form or suspension is preferably a mixture of solid particles of the compounds of formula (I) and formula (II) with a particle size of less than 200 μηι, the weight content of the compound of formula (1) relative to the weight of the compounds of formula (I). and (II) in said mixture between 0.5% and 20%, more preferably 0.5% and 10%.

The invention also relates to the aforementioned pharmaceutical compositions, which preferably comprise at least one pharmaceutically acceptable acid. Examples of such acids are: citric acid, glutamic acid, succinic acid, ethanesulfonic acid, acetic acid, tartaric acid, ascorbic acid, methanesulfonic acid, fumaric acid, adipic acid, malic acid and mixtures thereof. Citric acid is very suitable. The presence of said acid improves the stability of the active ingredient or ingredients.

The ratio of the weight of the pharmaceutically acceptable acid to the weight of said active solid particles is preferably between 0.01 and 0.5. even more preferably between 0.03 and 0.2. It is preferred that the amount of acid is sufficient to adjust the pH of the suspension between 2 and 6, more preferably between 3 and 5, most preferably between 3.5 and 4.5.

ΙΟ

Methods for preparing solid and liquid dosage forms of this pharmaceutical composition are described in WO / 95/28393, and these disclosures are incorporated herein by reference. These compositions preferably comprise a humectant and optionally a starch derivative as described in U.S. Patent 5,578,621, the contents of which are incorporated herein by reference with respect to the description of a possible humectant and starch derivatives. The humectant disclosed in U.S. Patent 5,578,621 serves as a dispersant.

These pharmaceutical compositions in solid dosage form, in liquid dosage form, such as pastes or ointments, may optionally contain other active agents such as antibiotics, antiviral agents or proton pump inhibitors. Although not preferred, these pharmaceutical compositions may also contain active solid particles of a compound of formula (I) and / or a compound of formula (II) of greater than 200 µm.

The compositions may contain excipients known to be useful in the preparation of forms suitable for oral administration.

Advantageously, in the interest of excellent activity against a wide variety of parasites, bacteria, fungi and viruses, the partition coefficient of said active solid particles is between 0.8 and 2, preferably between 1.1 and 1.9, most preferably higher than 1.5, wherein preferably the partition coefficient shall be calculated according to the formula:

F <> n% ⁼ (0911% - 0 10 ^.) / ((090% + 0,0, ..) / 2)

I

- Foo% is the partition coefficient at 90%;

Is the maximum particle size in the particle fraction corresponding to 90% of the said active solid particles, and

- 010%. is the maximum particle size in the particle fraction corresponding to 10% of said active solid particles.

In a specific embodiment of the invention, the particles of the compound of formula (I) and / or (II) are prepared by the above methods and are then milled so that less than 10% of said active particles exceed 100 μιη, less than% of said particles are greater than 50 μιη; less than 10% of said active particles are less than 5 μηι, the mean particle size being between 20 and 50 μιη. Thereafter, said active particles are granulated using a mixture comprising the active solid particles and at least one granulating agent. Examples of the granulating agent are: polyvinylpyrrolidone, water, alcohol, sucrose, hydroxycellulose and mixtures thereof. It is preferred that at least one pharmaceutically acceptable acid is added during the granulation process.

The invention relates to solid dosage forms comprising a composition of the invention such as tablets, dispersible tablets, coated tablets, matrices, and the like. The dosage form of the invention comprises, for example:

- solid active particles with a particle size below 200 μηι, with less than 10% of said particles having a size above 100 μιη, less than 50% of said particles having a size above 50 μιτι and less than 10% of said particles having a size below 5 μηι, a mean particle size of between 20 and 50 μηι;

at least one granulating agent;

- at least one humectant;

at least one starch derivative, and

at least one pharmaceutically acceptable acid, which is preferably added during the granulation process.

¹ I

Liquid dosage forms such as aqueous suspensions of a compound of the invention include, for example:

- as active agent, solid active particles comprising a compound of formula (I) and / or a compound of formula (11) having a particle size below 200 μιη, of which less than 10% of said particles are above 100 μιη, less than 50% of said particles are above 50 μιη and less than 10% of said particles have a size below 5 μιη a

at least one granulating agent;

- at least one humectant;

at least one pharmaceutically acceptable acid such that the pH of the suspension is between 2 and 6, preferably between 3 and 5, most preferably between 3.5 and 4.5;

at least one thickening agent, for example xanthan, aguar or carruba resin, crystalline cellulose, carboxymethylcellulose or mixtures thereof.

Pasty or ointment forms of the invention suitable for oral administration include, for example:

- as active agent, solid particles containing a compound of formula (I) and / or a compound of formula (II) with a particle size below 200 μιη, less than 10% of said particles having a size above 100 μιη, less than 50% of said particles having a size above 50 μιη and less than 10% of the said particles are less than 5

- at least one humectant;

at least one pharmaceutically acceptable acid such that the pH of the suspension is between 2 and 6, preferably between 3 and 5, most preferably between 3.5 and 4.5;

at least one thickening agent, for example xanthan, aguar or carruba resin, crystalline cellulose, carboxymethylcellulose or mixtures thereof.

Pastes or ointment forms for external or intravaginal administration include, for example:

and

- as an active agent, solid particles comprising a compound of formula (1) and / or a compound of formula (II) with a particle size below 200 μιη, less than 10% of said particles having a size above 100 μιη, less than 50% of said particles having a size above 50 μιη and less than 10% of said particles have a particle size of less than 5

- at least one humectant;

at least one pharmaceutically acceptable acid such that the pH of the suspension is between 2 and 6, preferably between 3 and 5, most preferably between 3.5 and 4.5;

cetyl alcohol and / or glyceride derivatives and / or propylene glycol;

I

I

at least one thickening agent, for example xanthan, aguar or carruba resin, crystalline cellulose, carboxymethylcellulose or mixtures thereof.

The dry and pure compound of formula (I) and the dry and pure compound of formula (II) were subjected to milling and screening.

After grinding, the particles of the compound of formula (I), the compound of formula (II), and mixtures thereof had a particle size distribution according to Figure 8. Figure 8 shows the percentage of particles having a smaller size than 0 µm.

It follows that:

less than 10% by weight of the particles have a particle size of less than about 5 μιη;

less than 10% by weight of the particles have a particle size of greater than about 70 μπι;

the mean particle size is about 40 µm;

- the particle coefficient is about 1.73, said particle coefficient being calculated according to the equation:

I

F <> 0% ⁼ (090 * 0% | O%) / ((0Q (W? I ⁺ 0, O%) / 2) wherein

- Fpd ·, is the partition coefficient at 90%;

Is the maximum particle size in the particle fraction corresponding to 90% of the said active solid particles, and

0m% is the maximum particle size in the particle fraction corresponding to 10% of said active solid particles.

The following tables illustrate examples of such compositions.

Table I

An example of an orodispersible tablet composition comprising, as active ingredients, a compound of formula (H) and a compound of formula (!)

Nitazoxanide (99%) + desacetylnitazoxanide (1%) 200 mg

Microcrystalline cellulose

Avicel pH 102 from FMC-USA 1 16 mg

Crospovidone 25 mg

Magnesium stearate 3 mg

Colloidal silica 5 mg

Citric acid 10 mg

Strawberry flavor no. 877720 from Robertet 10 mg

Sodium saccharate 2 mg

Table 2

An example of a composition of coated tablets for oral administration containing, as active ingredients, a compound of formula (II) and a compound of formula (I)

Nitazoxanide 500 mg

Corn starch 60 mg

Pregelatinised maize starch _t 70 mg

Hydroxypropylmethylcellulose 5 mg

Sucrose 20 mg

Sodium starch glycolate 30 mg

Citric acid 25 mg

Talc 8 mg

Magnesium stearate 7 mg

Coatings: hot sugar solution or coating film is sprayed onto tablets or granules containing 500 mg of the active ingredient

Table 3

An example of an aqueous suspension comprising a compound of formula (U) and a compound of formula (I) as active ingredients for oral administration; The pH of the suspension was about 4.1

Nitazoxanide (98%) + desacetylnitazoxanide (2%) 2 g

Distilled water 100 ml

Sodium benzoate 0.2 g

Sucrose 30.5 g

Xanthan resin 0.2 g

Microcrystalline cellulose and sodium carboxymethylcellulose

Avicel RC-59I from FMC-USA 0.8 g

Citric acid 0.2 g

Sodium citrate dihydrate 50 mg

Strawberry flavor no. 877720 from Robertet 125 mg

Red dye no. 33 D and C 1 mg

Table 4

An example of an oral paste containing a compound of formula (II) and a compound of formula (I) as active ingredients

Nitazoxanide (98%) + desacetylnitazoxanide (2%) 500 mg

Mineral oil 10 g

Raw sugar l 1 g

Microcrystalline cellulose and sodium carboxymethylcellulose

Avicel RC-591 from FMC-USA 0.8 g

Citric acid 0.2 g

Table 5

A paste or butter formulation for itilravagial or external administration, said paste or ointment comprising a compound of formula (II) and a compound of formula (I) as active ingredients

Nitazoxanide (98%) + desacetylnitazoxanide (2%) 8 g

Cremafor A6 2 g

Kremafor A25 1.5 g

Mineral oil 7 g

Luvitol EHO 7 g

Glyceryl monoester 4 g

Cetyl alcohol 3 g

Simethicone 0.5 g

Germaben II 1 g

Propylene glycol 3.5 g

Distilled water 62.5 g

The pharmaceutical compositions of the invention are compositions with a broad spectrum of effects on parasites, bacteria, fungi, and viruses, especially when administered orally.

The efficacy and safety of the above-described pharmaceutical compositions when administered to both animals and humans were excellent. In human clinical studies, it has been specifically found that the efficacy of the above pharmaceutical compositions in the treatment of parasitic diseases is significantly higher than that of the same formulations using an active compound with a particle size between 170 and 520 µm (mean particle size = 352 µm), although larger particles were administered to patients at doses up to three times higher and over a longer period. Examples of recovery rates are given in Table 6 below.

Table 6

Comparison of the results of clinical investigations in humans using the merger of Formula (I) and Formula (II) with particle sizes ranging from 70 μηι to 520 μηι (mean = 352 μηι) with results obtained using compounds of Formula (I) and Formula (II) with particle sizes in the range of 5 μηι to 200 μηι (mean = 34 μ / ijJ).

Compound of Formula (I) (98%) + Compound of Formula (10 (2%)

parasite Particle size 170 to 520 μιη Dose 15-50 mg / kg / day for 3 to 7 days Cure / Total =% Cured Particle size 5 to 200 μιη Dose 15 mg / kg / day for 3 days Cure / Total =% Cured Blastocystis hominis 12/27 = 44% 10/10 = 100% Entamoeba histolytica 29/47 = 63% 106/133 = 80% Giardia lamblia 1 1/37 = 30%. 50/73 = 68% Ascaris lumbricoides 3/69 = 4% 144/178 = 80% Trichuris trichiura 7/48 = 15% 57/79 = 73%

For all the parasites listed in Table 6, the recovery rate was significantly better in patients treated with active particles between 5 and 200 μηι than with active particles between 170 and 520 μιη, with in all cases a statistical significance of p <0.02 ( using standard tests X ² ). This was the case, although the doses of the active agent with a larger particle size were normally higher and the treatment time was often longer than the time of administration of the pharmaceutical compositions of the active agent with a particle size below 200 μιη. None of the patient groups experienced adverse reactions.

Results similar to those from human studies above were also found in animal tests

In addition, adverse reactions observed in dogs following oral administration of a single dose of 50 mg of compound of formula (1) and compound of formula (11) per kg dog weight were not observed in extensive animal studies using compound of formula (I) and compound of formula (II) particle sizes between 5 and 200 gm (mean> 10 gm), although the same dose or higher dose of these compounds was administered daily for 90 days or longer.

In addition, the compositions were still at 40 ° C and 65% relative humidity for six months or, in the case of liquid suspensions, when suspended in water under the above conditions, for 3 months to ensure that the active ingredients do not degrade and that the compositions maintain their effectiveness for a period of time after preparation that is suitable for therapeutic and commercial purposes.

The efficacy of the pharmaceutical compositions will be shown below.

DETAILED DESCRIPTION OF THE INVENTION

EXAMPLE 1

Cryptosporidium parvum

In a preparatory clinical experiment, 30 AIDS sufferers with chronic cryptosporidal diarrhea were treated by oral administration of nitazoxanide at daily doses of 500 to 2000 mg. If diarrhea persisted, nitazoxanide was continued for a further 4 weeks with a maximum daily dose of 2000 mg.

Twenty-eight subjects completed two or more weeks of treatment, and by the eighth week after treatment, 16 were able to evaluate for therapeutic response. In this latter group, 12 subjects showed a 50% or greater reduction in the daily frequency of excretion and ten individuals showed a significant reduction or complete elimination of parasites in the faeces, with four organisms undetectable. Six patients met the criteria for clinical and parasitological response.

Patients who received higher daily doses of the drug for a longer period of time had a better chance of a positive response.

A test with nitazoxanide in cryptosporidial diarrhea due to AIDS using a fluorescent label showed a decrease in the frequency of elimination in subjects who received 500, 1000, 1500 or 2000 mg of the drug daily. Participants showed CD4 + 42 cell / mm ³ counts (in the range of 0-303 cells / mm ³ ), an average daily stool count of 6.7 over 15 months, Cryptosporidium parvum oocysts in stool, and no other apparent intestinal pathogens. Almost all participants failed azithromycin or paromycin therapy.

At 23 weeks, 9 of 13 patients had a complete clinical response (predominantly shaped stools one to three times a day) and 4 of 13 had a partial clinical response (at least a 50% decrease in daily stool frequency or a change in stool consistency to at least 75% of shaped stools). By the end of the experiment, 8 out of 11 subjects had parasite complete extermination and a further 3 showed a significant decrease in oocyst concentration. There was a trend towards better response at doses of 1000 mg per day and above and for longer treatment. Three participants in the experiment received a hives rash on the skin; more than 90% of participants followed the health regime for more than four weeks.

EXAMPLE 2

Cryptosporidium parvum

In vitro experiment, dosing data:

Nitazoxanide was dissolved in sterile dimethylsulfoxide (DMSO) and tested on unicellular layers infected with intact Crypotosporidium parvum oocysts at concentrations of 100 µg / ml, 10 µg / ml, 1 µg / ml and 0.1 µg / ml. In parallel, a second test was performed at nitazoxanide concentrations of 20 pg / ml, 2 µg / ml, 0.2 µβ / ml and 0.02 µg / ml. These concentrations were achieved by a gradual dilution technique using complete DMEM broth until a final DMSO concentration of 0.5% was reached. The same dilution was achieved with the control medium (soil).

MDBKF5D2 cell culture grown in seven-millimeter chambers was used in the experiment; as Cyclosporidium parvum oocyst GCH1 at 5 x 10 ⁴ per well; it was a comparison of paromomycin (positive control) with nitazoxanide (experimental drug). Immune rabbit antisera containing Cryptosporidium parvum sporozoites (0.1%) and fluorescein-conjugated goat serum against rabbit antibody (1%) were also included.

Toxicity test:

200 μΐ of nitazoxanide containing broth at concentrations of 100, 10, 1 and 0.1 pg / ml and the respective control broths were plated in two wells of a 96 well plate containing confluent single-cell layers of MDBKF5D2 cells and two wells without this layer. The drug was incubated on one-cell layers at 37 ° C and 8% CO 2; after 24 hours (first experiment) and 48 hours (second experiment), MTS (Owen's solution) and PMS were added to each well at 333 µg / ml, respectively. 25 μΜ. The plate was returned to the incubator and developed in the dark for two hours. 100 μΐ of the supernatant was then removed from the culture and transferred to a new microtiter plate to determine the optical density by ELISA at 490 nm. The results were recorded and analyzed. Percentage toxicity was calculated by subtracting the mean optical density (OD) of the drug culture supernatant from the OD of the drug-free control soil supernatant, dividing by the mean optical density of the supernatant from the control soil and multiplying by 100.

OD of soil - OD of drug χ 100 OD of soil

Cryptosporidium parvum intact oocyst test

On confluent unicellular MDBKF5D2 layers, cryptosporidium parvum oocysts were incubated in nitazoxanide (100, 20, 10, 2, 1, 0.2, 0.1 and 0.02 pg / ml) at 37 ° C and 8% CO 2. x 10 ⁴ per well. The degree of infection in each well was determined by immunofluorescence analysis at 24 and 48 hours. Percent inhibition was determined by subtracting the mean number of parasites per 10 fields in test drug wells from the mean number of parasites per 10 fields in wells without drug control, dividing this difference by the mean number of parasites in control soils and multiplying by one hundred.

number in counter, soils - number in soils with medicine _x | qq number in control soils

The results:

Experiment 1: 24 hours

compound concentration Medium (+ SD) * toxicity % inhibition % Infected soil 0 983.5 (± 128.2) 0 0 paromomycin 2 mg / ml 482 (+47.1) 23.8 51 NTZ 100 g / ml loss 88.1 ON THE ** 10 μg / ml 55.5 (± 13.5) 65.1 94.4 1 μg / ml 224.5 (± 28.5) 8.3 77.2 OJ Mg / ml 474.5 (± 29.5) 19.3 51.8

* Number of parasites / 10 fields ** Not available due to toxicity

Experiment 2: 48 hours

compound concentration Medium (+ SD) * toxicity % inhibition % Infected soil 0 223 1.25 (+90.03) 0 0 paromomycin 2 mg / ml 580 (+33.42) 40.8 74,01 NTZ 20 μg / ml 68.75 (+13.77) 92.87 96,92 2 μg / ml 1 13.75 (+21.36) 24.93 94.90 0.2 μ§ / ηιΙ 1020 (+158.48) 16:56 54.29 0.02 pg / ml 1041 (+191.46) 21.23 c - » J J, J J

* Number of parasites / 10 fields

Effect of nitazoxanide on intact Cryptosporidium parvum oocysts

In Experiment 1, nitazoxanide at concentrations of 10, 1 and 0.1 μιη / ml resulted in parasite inhibition values of 94.4, 77.2 and 51.8%, and cell toxicity of 65.1, 8.3 and 19, respectively, 3%. Although dilution of 10 µg / ml showed almost complete inhibition of parasite infection, high levels of toxicity were evident. At a dilution of 1 µg / ml nitazoxanide, a comparison with paromomycin at a concentration of 2 mg / ml in favor of both parasite inhibition and cell toxicity (77.2% inhibition of parasites and 8.3% cellular toxicity for nitazoxanide at a dilution of 1 gg / ml compared favorably) with 51% inhibition of parasites and 23.8% cell toxicity for paromomycin at 2 mg / ml).

In Experiment 2, the drug was modified to achieve better dose distribution with minimal toxicity. As a result, the cultures remained alive after 48 hours and not only after 24 hours as in Experiment 1. Incubation for 48 hours, of course, resulted in higher relative cell toxicity than shown by comparison with paromomycin in both experiments. The nitazoxanide concentration of 20 µg / ml was still too toxic when incubated for 48 hours, although the single-cell layer appeared so far intact. It is possible that the high toxicity necessarily affecting cell function also has an effect on the development of parasite infection. At a dilution of 2 µg / ml nitazoxanide, the prevention of parasite infection was considerable at relatively low cellular toxicity. Other dilutions also resulted in significant inhibition and low cell toxicity. At a concentration of 2 μ§ / ιτι1, moderate cell toxicity and 94.90% inhibitory activity indicate that nitazoxanide at 2 µg / ml is more suitable than paromomycin in Cryptosporidium parvum infection in vitro than paromomycin at 2 mg / ml (1000 times) higher concentration).

EXAMPLE 3

Cryptosporidium parvum

Information: in vitro dosing and storage

Nitazoxanide and desacetylnitazoxanide (NTZ and NTZdes) were tested on unicellular layers infected with intact Cryplosporidium parvum oocysts and excited sporozoites at concentrations of 10, 1, 0.1 and 0.01 gg / ml. Each compound was dissolved in 100% dimethylsulfoxide (DMSO) and diluted with sterile DMEM medium. Each nitazoxanide concentration as well as control media contained a constant amount of 0.025% DMSO.

Cell cultures from MDBKF5D2 cells grown in seven millimeter wells were used in the experiment; GC.HI oocysts used at 5x10 ⁴ per well as cyclosporidium parvum; it was a comparison of paromomycin (positive control) against nitazoxanide (experimental drug). Immune rabbit antisera containing sporozoites (cryplosporid parvum (0.1%) and fluorescein-conjugated goat serum against rabbit antibody (1%) were also included.

Toxicity test:

200 μΙ of nitazoxanide-containing broth at the above concentrations and the respective control broths were plated in two wells of a 96 well plate containing a confluent single-cell layer of MDBKF5D2 cells and two wells without this layer. The drug was incubated on layers at 37 ° C with 8% CO 2; after 48 hours, MTS (Owen solution) and PMS were added to each well at 333 μβ / ηιΙ and 25 μΜ, respectively. The plate was returned to the incubator and developed in the dark for two hours. Then, 100 μΐ of each supernatant was removed from the culture and transferred to a new microtiter plate 11a to determine optical density by ELISA at 490 nm. The results were recorded and analyzed. Percent toxicity was calculated by subtracting the mean optical density (OD) of the drug supernatant from the mean OD of the drug-free control soil supernatant, dividing by the mean optical density of the supernatant from control soil and multiplying by 100.

Soil OD - OD drug

OD of soil x 100

The cytotoxicity classification was determined as follows: 0.5% toxicity = 0.6% to 25% toxicity = 1.26% to 50% toxicity = 2.5% to 75% toxicity = 3 and 76-100% toxicity = 4. that cytotoxicity 0 to 1 is to be considered as an acceptable level of toxicity. Toxicity values 2, 3 and 4 are considered to be highly toxic to the single cell layer.

Testing of intact Cryptosporidium parvum oocysts

On confluent unicellular MDBKF5D2 layers at 37 ° C and 8% CO 2, they were incubated in nitazoxanide at the above concentrations of Cryptosporidium parvum oocyst at 5 x 10 ⁴ per well. The degree of infection in each well was determined by immunofluorescence analysis after 48 hours and analyzed by computer. Percent inhibition was determined by subtracting the average number of parasites per field in the medicated wells from the average number of parasites per field in the drug-free well, dividing this difference by the average number of parasites in the control media and multiplying by 100.

number in contr. soils - number in soils with medicinal product χ 100 number in control soils

The results:

Experiment with Cryptosporidium parvum oocysts (48 hours)

drugs Horses. parasite ± SD Tox / O D ± SD Inhibited. % Tox. % classification Aqueous media 0 681.58 ± 271.02 2,024 ± 0.18 0 0 0 paromomycin 2000 115.75 ± 44.65 1,219 ± 0.009 83.02 39.79 2 DMSO the media 0 628.50 ± 171,94 1,799 ± 1.45 0 0 0 0,025 NTZ 10 11.75 ± 7.33 0,413 ± 0.13 98.13 77,07 4 1 39.67 ± 13.13 1,618 ± 0.326 93,69 10.09 1 , ⁰ ' ¹ 643.42 ± 229.73 1,878 ± 0.154 <0 <0 0 0.01 714.33 ± 194.79 1,617 ± 0.072 <0 10.12 1 New 10 13.75 ± 6.66 0,337 ± 0.005 97.81 81.27 4 NTZdes 1 39.92 ± 13.49 1,710 ± 0.033 93.65 4.97 0 0.1 649.86 ± 152.19 1,506 ± 0.119 <0 16.29 1 0.01 749.33 ± 139.49 1,721 ± 0.144 SO 4.36 0

Horses. pg / ml; Parasite - average number of parasites / field (12 fields analyzed); Inhibited. % - inhibition of parasite infection in%; Tox% - drug toxicity against cells in%.

From the above, it is clear that the inhibitory activity of NTZdes is the same as that of NTZ in Example II. Nitazoxanide and desacetylnitazoxanide were also effective in vitro against Cryptosporidium parvum in parallel tests, obtaining 98% and 94% inhibition results at dilutions of 10 and 1 µg / ml for both compounds. For nitazoxanide, a dilution of 1 µg / ml was the lowest concentration achieving greater than 90% inhibition, while 50% inhibition could be achieved with a lower nitazoxanide concentration of, for example, 0.2, 0.1 and 0.02 µg / L. Under the same experimental conditions, paromomycin used as a positive control was 2000 times less potent as it achieved an inhibitory potency of only 51-83% at a concentration of 2000 pg / ml.

EXAMPLE 4

E. intestinalis and V. cornea

2RK-I3 cells (rabbit liver cells) were plated on 24-well culture plates at 2.6 x 10 ⁵ cells per well (1.0 ml medium, RPMI 1640 with 2 mM L-glutamine and 5% heat activated bovine fetal cells). serum). Plates were incubated overnight in a CO 2 incubator at 37 ° C, during which time the wells were pooled (one doubling so that the number of cells per well was about 5 x 10 ⁵ ).

Seplata intestinalis (tissue culture) microorganisms were added to the host cells in an amount of about 3: 1, i.e. about 15 x 10 ⁶ microorganisms per well. As a result, about 50% of the host cells were infected.

Drugs were dissolved in DMSO, water or methanol (depending on solubility) to 1.0 mg / ml solutions. These solutions were stored at -70 ° C. All dilutions in these experiments were performed in complete tissue culture medium. All dilutions were assayed in three wells.

The medium (containing freshly diluted drugs) was replaced every three to four days.

On day 6 after addition of parasites and drugs, 11a cells were examined for toxicity. Control cells with drugs but without parasites are studied from several points of view: confluency, cell morphology and whether dead or floating cells are present. Cells incubated only with parasites are checked for parasite infectivity (presence of parasitophoric vacuoles). Cells incubated with parasites and drugs are evaluated for host cell toxicity and the amount of parasitophoric vacuoles present (whether large, medium, small)

On day 10, 100 μΙ 10% SDS (target concentration 0.5%) is added to the culture wells to rupture the host cell membranes and release microsporidia. The total amount of parasites in each well is determined by determining an aliquot with a hemacytometer. Results are expressed as percent inhibition (relative to number of infected cells without drug addition).

The results are shown in Figures 1 to 4.

EXAMPLE 5

Toxoplasma gondii

Nitazoxanide and desacetylnitazoxanide were tested against parasites, in particular against Toxoplasma gondii strain RH, maintained by repeated passages of mice. Cell cultures of MRC5 fibroplasts from Βίο-Merieux, France, were inoculated with this Toxoplasma gondii strain in 96-well culture plates. 200 freshly harvested tachyzoites were added to each culture well (except for 8 control wells used as negative control). After 4 hours of incubation, diluted drug solutions were added to these cultures.

Nitazoxanide (NTZ) and desacetylnitazoxanide (dNTZ) were tested at concentrations ranging between 8 x 10 ^-4 and 40 mg / L. Drugs were first dissolved in DMSO at concentrations of 2 mg / ml and then prepared at the necessary concentrations by successive dilutions in culture broth. No precipitation occurred.

These drug dilutions were added to the cultures (8 wells per dilution) followed by incubation of the culture plates for 72 hours. The cultures were then fixed with cold methanol. Toxoplasma gondii growth was detected by ELISA using a peroxidase labeled rabbit antibody to Toxoplasma gondii. The optical density was recorded for each well.

The results are presented in a graph plotting optical density data against drug concentration in culture. Statistical analysis consisted of regression analysis at a 95% confidence interval and determination of dose / response curves from the optical density values for each drug.

One plate was contaminated with Giemsa to determine its cytopathic effect on the cultures.

Three separate experiments were performed. Two culture plates were used for each compound in each experiment; In each culture plate, 8 replicate wells were used for each drug concentration.

The results

Similar results were obtained in three sets of experiments. A graphical representation of the results of one representative experiment for each drug is shown in Figures 5a, b, c and 6a, b, c.

Nitazoxanide (Figures 5a, b, c);

At concentrations of between 10 ^-4 mg / 1 and 0.3 mg / 1 did not show any inhibiting effect. Concentrations> 0.6 mg / L showed a significant effect and complete inhibition of Toxoplasmci growth was achieved at concentrations> 2.5 mg / L. However, at concentrations> 2.5 mg / L, significant toxicity was observed on the single cell layer.

Microscopic examination of the single-cell layer showed that NTZ at a concentration of 1.25 mg / l produced a cytopathic effect on the parasite-infected cells in the form of an increase in parasitophoric vacuoles and a decrease in the number of intracclular parasites. Based on regression analysis, it is estimated that a 50% inhibitory effect has a dilution of 1.2 mg / L

Desacetylnitazoxanid (Figures 7a, b, c):

The desacetyl Similar results were obtained: no effect for concentrations ranging from 10 ^-4 mg / 1 and 0.3 mg / 1, for inhibition of CII koncentrá29> 0.6 mg / l and marked toxicity for concentration> 2.5 mg / L. It can be estimated that a 50% inhibitory effect has a dilution of 1.2 mg / L.

The results obtained were reproducible in three separate experiments in evaluating the inhibitory effect of the drug on repeated cultures for each drug concentration.

Both NTZ and desacetylnitazoxanide showed significant inhibition of Toxoplcisma growth at concentrations of about 1.2 mg / l with changes to parasitophoric vacuoles, but without significant changes on the parasite itself.

These results show that these drugs have good activity against Toxoplasma gondii and that this therapeutic effect can be expected in vivo at concentrations of about 1 mg / l in serum or tissues.

EXAMPLE 6

Mycobacteria

Nitazoxanide has been found to have antimicrobial activity against mycobacteria of tuberculosis. The following table shows the results of the MIC detection of nitazoxanide and tizoxanide (NTZdes) against Mycobacterium incellular cell agar dilution technique. These results are based on several experiments using Middlebrook agar dilution technique, each of which lasted 3 weeks. The data obtained show that nitazoxanide has an MIC against Mycobacteria of 2 μg / ml and tizoxanide another MIC. 4 µg / ml using a standard strain of Mycobacterium intracelliilar from ATCC and a standard agar dilution technique.

Nitazoxanid MIC (minimal inhibitory endings of crayfish) against Mycobacteria intracellnlare

MIC * nitazoxanide 2 pg / ml tizoxanide 4 pg / ml

* MICs were determined by standard agar dilution technique for 3 weeks using Middlebrook 7HI 1 agar. M. intracellular ATCC 13950, a standard strain, was used in this experiment.

Figure 7 is a graph based on a study of nitazoxanide activity against mycobacteria grown in liquid broth. We used colorimetric analysis of MTS to determine growth after 4 hours compared to agar counting that allowed after 3 weeks. As can be seen in the data in Figure 7, when nitazoxanide was added 72 hours after the start of the culture, an immediate effect on the continuous growth compared to growth in the control medium alone. At a dose of 3 µg / ml nitazoxanide, growth stops for the next 24 hours and then slow growth continues for the next two days. The dose of 50 µg / ml was perfectly bacteriostatic during 144 hours of culture.

EXAMPLE 7

Cryptosporidium pnrvum

The effect of nitazoxanide against Cryptosporidium parvum was tested in experimentally infected mice. Nitazoxanide was supplied by Romark Laboratories, L.C. Tampa, Florida.

The total human dose (1 g / day for 7 days = 7 g) was modified for use in Paget and Barnes mice. The human dose was multiplied by a factor of 0.0026 for mice (weighing approximately 20 g) to obtain the total amount of drug required for each host in the morning and evening for 7 consecutive days. Each mouse received 2.6 mg daily (7000 mg x 0.0026 / 7). Doses were administered orally using a plastic syringe with a specially tipped needle.

Twenty (20) two-day-old suckling mice were infected by oral administration of 100,000 Cryptosporidium parvum oocysts obtained from infected calves. Prior to administration to mice, oocysts were concentrated using a sugar solution according to the technique described by Fayeroni and Ellis. Rectal swabs were collected from all mice and examined daily using the modified NiehlNeelsen contamination technique described by Graczyk et al. Oocysts appeared in the faeces 2 days after oral infection of the animals. From day 3 after infection of the animals, ten mice received 1.3 mg of nitazoxanide in the morning and evening for 7 consecutive days, while the 10 remaining mice were used as untreated control. Rectal swabs were collected daily on all seven days of treatment and every day for seven days following treatment discontinuation. Oocysts were suspended in oil and counted in 100 fields under a microscope.

The results:

The results shown in the following table clearly demonstrate that nitazoxanide administered at a daily dose of 2.6 mg / day for 7 consecutive days was effective against Cryptosporidium parvum and reduced the number of oocysts in the faeces of infected mice as compared to control animals. At the end of the third day of treatment, the test drug reduced the stool release in 6 out of 10 mice treated. On the seventh day at the end of treatment, this oocyst production was totally eliminated and all treated animals showed negative control results in droppings as opposed to untreated control mice. This effect lasted for at least 7 days after discontinuation of treatment following negative control results on the third and seventh day. treatment.

Number of oocysts / fields found in oil immersion Day 3 of treatment the last day of treatment Day 3 after treatment Day 7 after treatment Mouse # inspection gr. treated gr. inspection gr. treated gr. inspection gr. treated gr. inspection gr. treated gr. 1 3.0 0.0 5.0 0.0 4.0 0.0 2.0 0.0 2 4.0 0.0 4.0 0.0 3.0 0.0 1.0 0.0 3 6.0 0.0 5.0 0.0 4.0 0.0 0.5 0.0 4 3.0 2.0 3.0 0.0 2.0 0.0 1.0 0.0 5 5.0 2.0 3.0 0.0 3.0 0.0 0.5 0.0 6 3.0 0.0 4.0 0.0 5.0 0.0 2.0 0.0 7 3.0 0.0 5.0 0.0 4.0 0.0 1.0 0.0 8 5.0 1.0 5.0 0.0 1.0 0.0 0.5 0.0 9 3.0 3.0 3.0 0.0 2.0 0.0 1.0 0.0 10 0.0 5.0 0.0 2.0 0.0 0.5 0.0 Pretty 35 8.0 4.2 0.0 30 0.0 10 0.0 average 3.5 0.8 4.2 0.0 3.0 0.0 1.0 0.0 effectiveness 60% 100% 100% 100%

EXAMPLE 8

I

M ycob iicleri u m

Nitazoxanide was compared with the antibiotic isoniazid. BCG (Calmette and Guerin bacilli) was used as the mycobacterial strain. The sensitivity of this strain was the same as that of M. tuherculosis, but this strain was more harmless and therefore required no demanding measures.

Mice were given daily 4 mg of drug per mouse in 0.2 ml of sunflower oil. The results in nitazoxanide-treated mice were comparable to the isoniazid-treated group.

10 ⁷ 10 ⁵ spleen liver lungs spleen liver lungs Nitazo 1,575.000 1,575.000 57.500 68.250 70,000 50 800,000 1,550.000 122.500 65,000 87,500 75 875.000 1,550.000 30,000 75,000 35,000 150 950.000 750.000 75,000 60,000 60,000 50 1NH 475.000 1,050.000 11,000 20,000 21.250 50 255.000 750.000 5.750 15.250 27.500 125 200,000 975.000 4,000 60,000 20,000 52.500 37,500 50 50 PBS 1,500,000 2,125.000 92.500 102.500 195.000 750 1,525.000 1,800,000 98.000 140.000 175.000 800 1,925.000 1,750.000 177.500 98.000 150.000 500 1,675.000 1,800,000 117.500 105.000 150.000 750

EXAMPLE 9

Fascioln hcpntica

The efficacy of nitazoxanide and desacetylnitazoxanide against Fasciola hepatica was tested in vitro. , i

Adult Fasciola hepatica were obtained from liver bile ducts of three calves slaughtered for fasciolosis at Hardy's Meat Packers Veterinary Diagnostic Laboratory, Bunkie, LA The fluke was washed for one hour in sterile saline and transferred to sterile solution or RPMI (pH 7.4) for further 3 Hours. The fluke was then stored overnight at 37 ° C in 5% CO 2 in sterile RPMI-rabbit serum (50:50 volume ratio) or sterile RPMI (pH 7.4).

In vitro cultivation (37 ° C and 5% CO 2) was performed by modifying the method of Ibarra and Jenkins (Z. Parasitenkd. 70: 655-661, 1984). Under sterile conditions, the flukes were washed twice after 2-3 minutes in Hank's equilibrium saline (pH 7.2) and individually placed in six wells of Linbro culture plates containing aliquots of 10 ml of the intended dilution of the drug in the culture medium. This medium consisted of a mixture (in a volume ratio of 50-50) RPM1 and rabbit serum with 2% rabbit blood and 100 ppm penicillin and 100 ppm streptomycin. Only flukes with normal activity and morphology were used.

Stock solutions of NTZ or its metabolite D-NTZ from Romark were dissolved in DMSO (2000 pg / ml) and diluted in culture medium using a 100 ml volumetric flask to prepare a specific drug concentration (100, 50, 25, 10, 5). , 3.1 µg / ml). Two fluke were placed in each replicate culture, one in unsubstituted RBC culture medium and the other in drug-free and RBC culture medium.

The flukes were tested for drug effects such as death, movement disorders, or morphological changes compared to unmedicated control flukes using a backlit panel and illuminated three times the lens

The results:

I

Experiment 1:

In the case of D-NTZ, the fluke at 50 and 100 gg for one hour were dead or dying. At a concentration of 25 µg, 4 of the 7 fluke were dying after the first hour, two were active and one was malaise; within three hours they were all dead except two malaise and only one fluke remained malaise after four hours. At a drug concentration of 10 µg, limited activity was noted after 1, 3 and 4 hours and all were dead or dying within 7 hours. At the 5 µg and 3 µg dilutions, some individuals were observed to have decreased activity, with a slightly weaker onset in the 3 µg dilution category; in the 3 and 5 µg groups, all fluke were dead within 50 hours except one sluggish fluke in each group In the 1 µg group, activity reduction was observed after 42-74 hours and only 3 active and one remained active after 91 hours dying inotolica; in this group I pg, only one malaise fluke remained after 115 hours. In the RBC control group, mortality was monitored at 66 hours (one fluke), 91 hours (one fluke) and 15 hours (four fluke). In the non-RBC control group, all flukes were alive after 91 hours and one was dead at 115 hours.

Experiment 2:

In NTZ, a slightly greater activity was observed in 8 replicated cases compared to D-NTZ by an earlier effect on momentum and mortality. In the 100, 50, and 25 pg drug groups, all fluke except one in the 25 pg group, which was dead after three hours, were dead or dying after one hour. In all other treatment groups, there was a marked dose-dependent decrease in momentum after one hour. In the 10 µg group, only one fluke survived after 16 hours. In the 5 µg group, only 3 fluke were active after 6 hours and none was active after 16 hours. In the 3 µg group, only 2 malaise fluke were alive after 23 hours; but these were dead by 4l'hodinách In Group 1 pg died within 16 hours, one possible _apos alfalfa, within 41 hours of the three flukes and five by hour 74; 3 fluke were active after 91 hours and one fluke retained activity after 115 hours In the control group with RBC, 7 out of 8 fluke were active after 74 hours, after 91 hours 3 and two fluke remained active after II 5 hours In a non-RBC control group after 74 hours of active 6 out of 8 fluke, after 91 hours of 4 and two fluke remained active after 1115 hours.

In the high dose groups (25, 50, 100 µg), fluke death was rapid and was associated with contraction and curling of the ventral portion. At low concentrations, most fluke slowed down in time and was more sober and flattened after death or dying. Starting at 91 hours, contamination has been a limiting factor in the results of the experiments in some replicated cases. In the case of D-NTZ, a continuous overlap of two replicated plates with bacterial or fungal growth followed by mortality occurred after 115 hours. In the NTZ experiment, this overlap and truncated efficacy on whole replicated plates occurred after 91 hours (two replicated plates) and 115 hours (5 replicated plates). Observation after 139 hours was not considered valid due to total contamination of most plates.

conclusions:

Experiments with both investigational drugs have shown their considerable efficacy in killing fluke. A slightly higher potency against F. hepatica has been observed with nitazoxanide than with desacetylnitazoxanide, its major metabolite, which is considered to be effective at the hepatic level.

Rapid fluke death occurs with in vitro application of D-NTZ at concentrations> 50 µg per hour, at 25 µg for 4 hours, and at 10 µg for 6-7 hours. Ten µg may be a suitable single treatment targeted dose of the drug as long as pharmacokinetic data indicate that the tissue concentration is maintained> 6-8 hours after the single treatment.

Strong killer fluke killing within 74 hours (three days) was found for both compounds at concentrations of 3 µg and 5 µg, respectively. Longer survival, but not as long as the control unmedicated fluke, was observed at a concentration of 1 µg; therefore, the use of this drug at this concentration on fluke in the liver for 3 to 4 days may therefore have an insufficient effect on parasites.

EXAMPLE 10

Fascioln giganticn

Nitazoxanide was tested for efficacy against juvenile and adult Fasciola giganlica in experimentally infected rabbits.

Encystic Fasciola giganlica (EMC) metacercarcans were collected on cellophane film 28 to 35 days after slug infection Ĺ. calludi miracidia Fasuola giganlica using the technique described by Abdel-Ghana, in which slugs are exposed to artificial light for 30 minutes in pure, de-chlorinated tap water. The obtained encysted metacercariae (EMC) were stored for 5 to 8 days at 4 ° C in a refrigerator under water before they were used to infect experimental animals.

Forty (40) Boscat rabbits weighing 1.5 to 2 kg were used in this study and were assigned to two experimental groups of 20 individuals each.

Group 1 animals were orally infected with 35-40 encysted metacercaria wrapped in a lettuce leaf inserted at the root of the animal tongue. The animal paws were hand held closed until encysted metacercaria were swallowed. This group 1 was used to test nitazoxanide against immature stages (4-5 weeks old) of Fasciola giganlica.

Group 2 animals were orally infected as in the first group with 10-15 encysted metacercariae and used to test the efficacy of nitazoxanide against fluke in early adulthood (> 10 weeks old).

Ten animals in Group 1 were given 5 mg of nitazoxanide at 7 consecutive days in the morning and in the evening 4 weeks after their infection with the parasites in the early stage of their developmental cycle. The remaining 10 animals of Group 1 formed the untreated control group.

Ten animals in Group 2 were given 35 mg of nitazoxanide at 7 consecutive days in the morning and in the evening, 10 weeks away from their parasite infection in the adult stage of their developmental cycle. The remaining 10 animals of Group 2 formed the untreated control group.

All animals were fed dry food until the end of the experiment.

Seven days after the last nitazoxanide dose, all animals in both groups were sacrificed. The surface of the liver was examined for the presence of migrating necrotic grooves especially for the developmental stages of juvenile parasites. These necrotic regions were studied using two surgical needles in order to obtain migratory juvenile flukes using the technique described by El-Bahy. The liver was cut into small pieces, especially around the migrating grooves, and macerated under a microscope to extract flukes. The abdominal cavity and visceral surfaces were washed with warm water. This rinse water was combined, passed through a sieve, and examined for the presence of juvenile fluke. All parasites found, including their body parts, were counted in treated and untreated animals in both Groups 1 and 2. Live flukes were light pink in color, translucent and intact, washed away easily from the liver tissues with warm water while dead flukes were color greyish, they were relaxed and exhibited damaged necrotic surfaces. The activity of nitazoxanide was calculated using the formula:

% efficiency = a - b x 100 and where: a = number of fluke obtained from the faeces of control animals;

b = number of fluke obtained from the faeces of treated animals.

The results:

The research results presented in Table 7 show a significant decrease in the number of juvenile fluke extracted from the rabbit liver of the treated group compared to the control group. This percentage drop was 46.77% on average (ranging from 40 to 60%).

Table 7:

Efficacy of nitazoxanidia against juveniles (4 weeks old) Fasciola gigantica in experimentally infected rabbits

Number of fluke obtained from rabbit liver Rabbit no. Untreated control Treated rabbits Efficiency% 1 7 4 42% 2 7 4 42% J 6 3 50% 4 8 4 50% 5 5 3 40% 6 5 2 60% 7 5 3 40% 8 · 6 3 50% 9 8 4 50% 10 5 3 40% average 6.2 3.3 46,77%

In the early stages of early adulthood fluke in which rabbits were infected, nitazoxanide showed total efficacy (100% reduction) and no worms (fluke) were observed in the liver of treated rabbits compared to animals from the untreated rabbit control group, as shown in Table 8. .

Table 8 (/ nitazoxanide activity was laundered in early adulthood (10 weeks old) Fasciota giganlica in experimentally infected rabbits).

Number of fluke obtained from rabbit liver

Rabbit no. Untreated control Treated rabbits Efficiency% 1 4 0.0 100% 2 4 0.0 100% 3 J 0.0 100% 4 3 0.0 100% 5 2 0.0 100% 6 2 0.0 100% 7. 2 0.0 100% 8 3 0.0 100% 9 3 0.0 100% 10 3 0.0 100% average 2.9 0.0 100%

Nitazoxanide administered for 7 consecutive days at a dose of 70 mg / day is moderately effective against juveniles of Fasciota giganlica and completely effective against these parasites in the early adulthood.

EXAMPLE 13

Schistosoma

Nitazoxanide was tested against Schistosoma mansoni and Schistosoma hematohium in experimentally infected mice.

Forty (40) white mice weighing 30-50 g were divided into two treatment groups of 20 animals each. The first group was infected with 300,500 free and active Schistosoma mansoni cercariae suspended in

0.25 ml of distilled water and intraperitoneal injection given to all mice The second group was infected in the same way but with cerebral Schislosama hematohium. Both groups were then kept in the laboratory for a total of 70 days.

Seventy days after infection of the animals, each group of 10 animals treated with nitazoxanide at a dose of 1.3 ing administered for seven consecutive days orally in the morning and evening. Seven days after treatment, all animals were sacrificed and worms were extracted from the liver of each mouse by perfusion with lukewarm water (37 ° C). Schistosoma individuals obtained in all treated and control animals were counted. Nitazoxanide potency was calculated using the following formula:

% efficiency = 3 - ~ b x 100 a

where: a = number of Schistosoma individuals obtained from the washings of control animals b = number of Schistosoma individuals obtained from the washings of treated animals.

The results:

The results in Tables 9 and 10 clearly demonstrated that nitazoxanide administered at doses of 2.6 mg / day for 7 consecutive days was more effective against Schistosoma hematohium, where a reduction in worms of 82.85% compared to control animals was observed than against Schistosoma maiisoiii, where the reduction against the control group of mice reached only 59.91%. These results are consistent with those of Abaza et al., According to which nitazoxanide was not effective in treating patients against S. mansoni, as evidenced by a positive egg finding after nitazoxanide treatment.

Table 9

Efficacy of nitazoxanide against adults (33 weeks old) Schisloso ma mansoni in mice.

Number of fluke removed from mouse liver Mouse # Untreated control mice Treated mice 1 21 10 2 29 9 J 32 10 4 26 1 1 5 24 13 6 19 10 7 20 9 8. 24 12 9 22 8 10 30 7 Pretty 247 99 Diameter / mouse 24.7 9.9 effectiveness 59.91

Table 10

Efficacy of nitazoxanide against adult (13 weeks old) Schistoso has haem / obinm in mice.

Number of fluke removed from mouse liver Mouse # Untreated control mice Treated mice 1 18 3 2 16 Λ J 3 14 2 4 19 2 5 12 4 6 10 4 7th 13 2 8 12 2 9 17 0.0 10 9 2 Pretty 140 24 Diameter / mouse 14 2.4 effectiveness 82.85

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Claims (22)

PATENT CLAIMS A pharmaceutical composition for oral administration comprising, as an active ingredient, at least one compound selected from the group consisting of: compound of formula (I) Composition according to claim 1, characterized in that said active compound is in the form of active particles with a particle size of less than 200 gm and a mean particle size of greater than 10 gm Composition according to claim 2, characterized in that the average particle size of said active particles is between 10 and 100 gm. Composition according to claim 2, characterized in that the mean particle size of said active particles is between 20 and 50 gm. The composition of claim 2, wherein less than 10% by weight of said active particles have a particle size greater than 100 µηι. The composition of claim 2, wherein at least 50% by weight of said active particles have a particle size of less than 50 µηι. The composition of claim 2, wherein less than 10% by weight of said active particles have a particle size of less than 5 µηι. The composition of claim 2, wherein said composition comprises a mixture of active particles of compounds of formula (I) and formula (II), wherein the weight content of the compound of formula (I) is relative to the weight of the compounds of formula (I) and formula (II). II) in said mixture between 0.5 and 20%. The composition of claim 1, wherein said composition further comprises at least one pharmaceutically acceptable acid. The composition of claim 9, wherein said pharmaceutically acceptable acid is selected from the group consisting of citric acid, glutamic acid, succinic acid, ethanesulfonic acid, acetic acid, tartaric acid, ascorbic acid, methanesulfonic acid, fumaric acid, adipic acid, malic acid and mixtures thereof. The composition of claim 9, wherein said active ingredient is in the form of solid particles having a particle size of less than 200 gm and a mean particle size of greater than 10 gm, and wherein the weight ratio of pharmaceutically acceptable acid / weight of said solids is between 0.01 and 0.5. The composition of claim 11, wherein the weight ratio of pharmaceutically acceptable acid / weight of said solid particles is between 0.03 and 0.2. Use of a pharmaceutically acceptable composition comprising, as an active ingredient, at least one compound selected from the group consisting of a compound of formula (I) for the manufacture of a medicament for treating immunocompromised mammals infected with a microorganism selected from the group consisting of Cryptosporidium parvum, Isospora belli, Enterocytzoon bineusi, Encephalitozoon inteslinalis, Mycobacterium tuberculosis, Mycobacterium avium intracellulare, Pneumocystis carinii and Toxoplasma gondii. Use of a pharmaceutical composition according to claim 13, characterized in that said active ingredient is in the form of particles with a mean particle size of between 10 and 200 µmη. Use of a pharmaceutical composition according to claim 13, characterized in that said active ingredient is in the form of particles with a mean particle size of between 20 and 50 µηι. Use of a pharmaceutical composition according to claim 13, characterized in that said pharmaceutical composition comprises at least one pharmaceutically acceptable acid. Use of a pharmaceutical composition according to claim 16, characterized in that said pharmaceutically acceptable acid is selected from the group consisting of citric acid, glutamic acid, succinic acid, ethanesulfonic acid, acetic acid, tartaric acid, ascorbic acid, methanesulfonic acid, acid. fumaric acid, adipic acid, malic acid and mixtures thereof. I Use of a pharmaceutical composition according to claim 13, characterized in that said active ingredient is a compound of formula (I). Use of a pharmaceutical composition according to claim 13, characterized in that said active ingredient is a compound of formula (II). Use of a pharmaceutical composition according to claim 13, characterized in that said mammal is a human and that said active ingredient is administered in an amount of from 500 to 2000 mg per day. Use of a pharmaceutical composition according to claim 20, characterized in that said active ingredient is administered in an amount of from 1000 to 1500 mg per day. Use of a pharmaceutical composition comprising as an active ingredient at least one compound selected from the group consisting of a compound of formula (I)